DNA sequencing generally involves the generation of four populations of single-stranded DNA fragments having one defined terminus and one variable terminus. The variable terminus generally terminates at specific nucleotide bases (either guanine (G), adenine (A), thyminine (T), or cytosine (C)). The four different sets of fragments are each separated on the basis of their length, one procedure being on a high resolution polyacrylamide gel; each band on the gel corresponds to a specific nucleotide in the DNA sequence, thus identifying the positions in the sequence.
A frequently used DNA sequencing method is the dideoxy or chain-terminating sequencing method, which involves the enzymatic synthesis of a DNA strand (Sanger et al., 1977, Proc. Natl. Acad. Sci. USA 74:5463, which is incorporated herein by reference). Four separate syntheses are generally run, each reaction being caused to terminate at a specific base (G, A, T, or C) via incorporation of an appropriate chain-terminating nucleotide, such as a dideoxynucleotide. The reaction products are easy to interpret since each lane corresponds only to either G, A, T, or C.
In the dideoxy chain-terminating method a short single-stranded primer is annealed to a single-stranded template. The primer is elongated at its 3' end by the incorporation of deoxynucleotides (dNTPs) until a dideoxynucleotide (ddNTP) is incorporated. When a ddNTP is incorporated, elongation ceases at that base. However, to assure fidelity of DNA replication, DNA polymerases have a very strong bias for incorporation of their normal substrates, dNTPs, and against incorporation of nucleotide analogues, referred to as unconventional nucleotides. In the case of DNA synthesis, ribonucleotides (rNTPs) are considered unconventional nucleotides, because, like ddNTPs, rNTPs are not the normal in vivo substrate of a DNA polymerase. In the cell this property attenuates incorporation of abnormal bases such as deoxyinosine triphosphate (dITP) or rNTPs in a growing DNA strand.
Two frequently used automated sequencing methodologies are dye-primer and dye-terminator sequencing. These methods are suitable for use with fluorescent label moieties. Although sequencing can also be done using radioactive label moieties, fluorescence-based sequencing is increasingly preferred. Briefly, in dye-primer sequencing, a fluorescently labeled primer is used in combination with unlabeled ddNTPs. The procedure requires four synthesis reactions and up to four lanes on a gel for each template to be sequenced (one corresponding to each of the base-specific termination products). Following primer extension, the sequencing reaction mixtures containing dideoxynucleotide-incorporated termination products routinely are electrophoresed on DNA sequencing gel. Following separation by electrophoresis, the fluorescently-labeled products are excited with a laser at the bottom of the gel and the fluorescence is detected with an appropriate monitor. In automated systems, a detector scans the bottom of the gel during electrophoresis, to detect whatever label moiety has been employed, as the reactions pass through the gel matrix (Smith et al., 1986, Nature 321:674-679, which is incorporated herein by reference). In a modification of this method, four primers are each labeled with a different fluorescent marker. After the four separate sequencing reactions are completed, the mixtures are combined and the reaction is subjected to gel analysis in a single lane, and the different fluorescent tags (one corresponding to each of the four different base-specific termination products) are individually detected.
Alternatively, dye-terminator sequencing methods are employed. In this method, a DNA polymerase is used to incorporate dNTPs and fluorescently labeled ddNTPs onto the growing end of a DNA primer (Lee et al., 1992, Nucleic Acid Research 20:2471). This process offers the advantage of not having to synthesize dye-labeled primers. Furthermore, dye-terminator reactions are more convenient in that all four reactions can be performed in the same tube. Modified thermostable DNA polymerases having reduced discrimination against ddNTPs have been described (see European Patent Application, Publication No. EP-A-655 506 and U.S. patent application Ser. No. 08/448,223). An exemplary modified thermostable DNA polymerase is the mutated form of the DNA polymerase from T. aquaticus having a tyrosine residue at position 667 (instead of a phenylalanine residue), i.e. is a so called F667Y mutated form of Taq DNA polymerase. AmpliTaq.RTM. FS, manufactured by Hoffmann-La Roche and marketed through Perkin Elmer, reduces the amount of ddNTP required for efficient nucleic acid sequencing of a target by hundreds to thousands-fold. AmpliTaq.RTM. FS is a mutated form of the DNA polymerase from T. aquaticus having the F667Y mutation and additionally an aspartic acid residue at position 46 (instead of a glycine residue; G46D mutation).
There is a need for thermostable DNA polymerases that enable alternative nucleic acid synthesis methods for accurate and cost effective nucleic acid DNA sequence analysis. Fluorescence-based methods that do not require the use of dideoxynucleotides would be desirable. The present invention addresses these needs.